title: Extracellular alpha galactosidase (E.C. 3.2.1.22) from Aspergillus ficuum NRRL 3135 purification and characterization.
abstract: Extracellular alpha-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by ion exchange and hydrophobic interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 X10(4) M-1 cm-1. The purified enzyme was remarkably stable at 0 degrees C. It had a broad temperature optimum and maximum catalytic activity was at 60 degrees C. It retained 33% of its activity after 10 min. at 65 degrees C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The Kms for p-nitrophenyl-alpha-D-galactopyranoside, o-nitrophenyl-alpha-D-galactopyranoside and m-nitrophenyl-alpha-D-galactopyranoside are: 1462, 839 and 718 microM. The enzyme was competitively inhibited by mercury (19.8 microM), silver (21.5 microM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).
authors.affiliation: Department of Molecular Biology and Microbiology, University of Central Florida, Orlando 32816.
authors.forename: I G
authors.initials: IG
authors.lastname: Zapater
authors.forename: A H
authors.initials: AH
authors.lastname: Ullah
authors.forename: R J
authors.initials: RJ
authors.lastname: Wodzinski
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D001667:Binding, Competitive; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005285:Fermentation; D005696:Galactosidases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013696:Temperature
chemical_list: D005696:Galactosidases